Exosomes derived from human dermal fibroblasts (HDFn-Ex) alleviate DNCB-induced atopic dermatitis (AD) via PPARα.

Atopic dermatitis (AD) is a chronic inflammatory skin disease. Skin barrier dysfunction is the initial step in the development of AD. Recently, exosomes have been considered as potential cell-free medicine for skin defects such as aging, psoriasis and wounds. The aim of this study was to investigate the effects of human dermal fibroblast-neonatal-derived exosome (HDFn-Ex) on AD. HDFn-Ex increased the expression of peroxisome proliferator activated receptor α (PPARα) and alleviated the 1-chloro-2,4-dinitrobenzene (DNCB)-mediated downregulation of filaggrin, involucrin, loricrin, hyaluronic acid synthase 1 (HAS1) and HAS2 in human keratinocyte HaCaT cells. However, these effects were inhibited by the PPARα antagonist GW6471. In the artificial skin model, HDFn-Ex significantly inhibited DNCB-induced epidermal hyperplasia and the decrease in filaggrin and HAS1 levels via a PPARα. In the DNCB-induced AD-like mouse model, HDFn-Ex administration reduced epidermis thickening and mast cell infiltration into the dermis compared to DNCB treatment. Moreover, the decreases in PPARα, filaggrin and HAS1 expression, as well as the increases in IgE and IL4 levels induced by DNCB treatment were reversed by HDFn-Ex. These effects were blocked by pre-treatment with GW6471. Furthermore, HDFn-Ex exhibited an anti-inflammatory effect by inhibiting the DNCB-induced increases in IκBα phosphorylation and TNF-α expression. Collectively, HDFn-Ex exhibited a protective effect on AD. Notably, these effects were regulated by PPARα. Based on our results, we suggest that HDFn-Ex is a potential candidate for treating AD by recovering skin barrier dysfunction and exhibiting anti-inflammatory activity.

Systemic Type 2 Inflammation-Associated Atopic Dermatitis Exacerbates Periodontitis.

INTRODUCTION: Atopic dermatitis (AD) is a prevalent and chronic inflammatory skin disease characterized by Th2 cell-mediated type 2 inflammation. Emerging evidence indicated that AD patients exhibit an increased incidence of oral disorders. In the present study, we sought mechanistic insights into how AD affects periodontitis. METHODS: Onset of AD was induced by 2,4-dinitrochlorobenzene (DNCB). Furthermore, we induced periodontitis (P) in AD mice. The effect of AD in promoting inflammation and bone resorption in gingiva was evaluated. Hematoxylin and eosin staining, tartrate-resistant acid phosphatase staining, immunofluorescence assay, and flow cytometry were used to investigate histomorphology and cytology analysis, respectively. RNA sequencing of oral mucosa is used tissues to further understand the dynamic transcriptome changes. 16S rRNA microbial analysis is used to profile oral microbial composition. RESULTS: Compared to control group, mice in AD group showed inflammatory signatures and infiltration of a proallergic Th2 (Th2A)-like subset in oral mucosa but not periodontitis, as identified by not substantial changes in mucosa swelling, alveolar bone loss, and TRAP+ osteoclasts infiltration. Similarly, more Th2A-like cell infiltration and interleukin-4 levels were significantly elevated in the oral mucosa of DNCB-P mice compared to P mice. More importantly, AD exacerbates periodontitis when periodontitis has occurred and the severity of periodontitis increased with aggravation of dermatitis. Transcriptional analysis revealed that aggravated periodontitis was positively correlated with more macrophage infiltration and abundant CCL3 secreted. AD also altered oral microbiota, indicating the re-organization of extracellular matrix. CONCLUSIONS: These data provide solid evidence about exacerbation of periodontitis caused by type 2 dermatitis, advancing our understanding in cellular and microbial changes during AD-periodontitis progression.

Selective activation of TRPA1 ion channels by nitrobenzene skin sensitizers DNFB and DNCB.

2, 4-dinitrofluorobenzene (DNFB) and 2, 4-dinitrochlorobenzene (DNCB) are well known as skin sensitizers that can cause dermatitis. DNFB has shown to more potently sensitize skin; however, how DNFB and DNCB cause skin inflammation at a molecular level and why this difference in their sensitization ability is observed remain unknown. In this study, we aimed to identify the molecular targets and mechanisms on which DNFB and DNCB act. We used a fluorescent calcium imaging plate reader in an initial screening assay before patch-clamp recordings for validation. Molecular docking in combination with site-directed mutagenesis was then carried out to investigate DNFB and DNCB binding sites in the TRPA1 ion channel that may be selectively activated by these tow sensitizers. We found that DNFB and DNCB selectively activated TRPA1 channel with EC50 values of 2.3 ± 0.7 µM and 42.4 ± 20.9 µM, respectively. Single-channel recordings revealed that DNFB and DNCB increase the probability of channel opening and act on three residues (C621, E625, and Y658) critical for TRPA1 activation. Our findings may not only help explain the molecular mechanism underlying the dermatitis and pruritus caused by chemicals such as DNFB and DNCB, but also provide a molecular tool 7.5-fold more potent than the current TRPA1 activator allyl isothiocyanate (AITC) used for investigating TRPA1 channel pharmacology and pathology.

Anti-Atopic Dermatitis Effect of TPS240, a Novel Therapeutic Peptide, via Suppression of NF-κB and STAT3 Activation.

Atopic dermatitis (AD) is a relapsing skin disease with persistent inflammation as a causal factor for symptoms and disease progression. Current therapies provide only temporary relief and require long-term usage accompanied by side effects due to persistent relapses. A short peptide, TPS240, has been tested for its potential to subside AD. In this study, we confirmed the anti-atopic effect of TPS240 in vivo and in vitro using a DNCB-induced AD mouse model and TNF-α/IFN-γ-stimulated HaCaT cells. In the AD mouse model, topical treatment with TPS240 diminished AD-like skin lesions and symptoms such as epidermal thickening and mast cell infiltration induced by DNCB, similar to the existing treatment, dexamethasone (Dex). Furthermore, skin atrophy, weight loss, and abnormal organ weight changes observed in the Dex-treated group were not detected in the TPS240-treated group. In TNF-α/IFN-γ-stimulated HaCaT cells, TPS240 reduced the expression of the inflammatory chemokines CCL17 and CCL22 and the pruritic cytokines TSLP and IL-31 by inhibiting NF-κB and STAT3 activation. These results suggest that TPS240 has an anti-atopic effect through immunomodulation of AD-specific cytokines and chemokines and can be used as a candidate drug for the prevention and treatment of AD that can solve the safety problems of existing treatments.

Vasicine alleviates 2,4-dinitrochlorobenzene-induced atopic dermatitis and passive cutaneous anaphylaxis in BALB/c mice.

Atopic dermatitis (AD), a type of skin inflammation, is associated with immune response mediated by T-helper 2 (Th2) cells, and mast cells. Vasicine is an alkaloid isolated from Adhatoda vasica, a popular Ayurvedic herbal medicine used for treating inflammatory conditions. In the present study, the anti-AD effects of vasicine were evaluated on 2,4-dinitrochlorobenzene-induced AD-like skin lesions in BALB/c mice. The potential anti-allergic effects of vasicine were also assessed using the passive cutaneous anaphylaxis (PCA) test. The results showed that the oral administration of vasicine improved the severity of AD-like lesional skin by decreasing histopathological changes and restoring epidermal thickness. Vasicine also inhibited the infiltration of mast cells in the skin and reduced the levels of pro-Th2 and Th2 cytokines as well as immunoglobulin E in the serum. Finally, vasicine inhibited the expression of pro-Th2 and Th2 cytokines in skin tissues, indicating the therapeutic potential of vasicine for AD.

The anti-inflammatory properties of ethyl acetate fraction in ethanol extract from Sarcodia suiae sp. alleviates atopic dermatitis-like lesion in mice.

Atopic dermatitis (AD) is a chronic inflammatory and pruritic disease; it can be treated by inhibiting inflammation. Sarcodia suiae sp. is an edible, artificially cultivable red algae with multiple bioactivities. We assessed the anti-inflammatory activity of the ethyl acetate fraction of S. suiae sp. ethanol extract (PD1) on 1-chloro-2,4-dinitrochlorobenzene (DNCB)-induced AD-like lesions. Results show that PD1 alleviated symptoms and significantly decreased clinical dermatitis score. PD1 inhibited serum immunoglobulin E expression and alleviated swelling in the spleen and subiliac lymph nodes. In skin tissues, PD1 alleviated aberrant hyperplasia, decreased epidermal thickness, and decreased the accumulation of mast cells. PD1 mediated the recovery of skin barrier-related proteins, such as claudin-1 and filaggrin. Our study demonstrated that PD1 has anti-inflammatory effects, alleviates AD symptoms, inhibits inflammatory responses in skin tissues, and restores barrier function in DNCB-induced AD mice. These findings reveal that S. suiae sp. extract provides an alternative protective option against AD.

Protective Effects of Topical Administration of Laminarin in Oxazolone-Induced Atopic Dermatitis-like Skin Lesions.

Laminarin is a polysaccharide isolated from brown marine algae and has a wide range of bioactivities, including immunoregulatory and anti-inflammatory properties. However, the effects of laminarin on atopic dermatitis have not been demonstrated. This study investigated the potential effects of topical administration of laminarin using a Balb/c mouse model of oxazolone-induced atopic dermatitis-like skin lesions. Our results showed that topical administration of laminarin to the ear of the mice improved the severity of the dermatitis, including swelling. Histological analysis revealed that topical laminarin significantly decreased the thickening of the epidermis and dermis and the infiltration of mast cells in the skin lesion. Serum immunoglobulin E levels were also significantly decreased by topical laminarin. Additionally, topical laminarin significantly suppressed protein levels of oxazolone-induced proinflammatory cytokines, such as interleukin-1ß, tumor necrosis factor-α, monocyte chemoattractant protein-1, and macrophage inflammatory protein-1α in the skin lesion. These results indicate that topical administration of laminarin can alleviate oxazolone-induced atopic dermatitis by inhibiting hyperproduction of IgE, mast cell infiltration, and expressions of proinflammatory cytokines. Based on these findings, we propose that laminarin can be a useful candidate for the treatment of atopic dermatitis.

Topical Melatonin Exerts Immunomodulatory Effect and Improves Dermatitis Severity in a Mouse Model of Atopic Dermatitis.

Oral melatonin supplement has been shown to improve dermatitis severity in children with AD, but the mechanism of the effect is unclear, and it is uncertain whether melatonin has a direct immunomodulatory effect on the dermatitis. Topical melatonin treatment was applied to DNCB-stimulated Balb/c mice, and gross and pathological skin findings, serum IgE, and cytokine levels in superficial lymph nodes were analyzed. Secretion of chemokines and cell proliferative response after melatonin treatment in human keratinocyte HaCaT cells were also studied. We found that in DNCB-stimulated Balb/c mice, topical melatonin treatment improved gross dermatitis severity, reduced epidermal hyperplasia and lymphocyte infiltration in the skin, and decreased IP-10, CCL27, IL-4, and IL-17 levels in superficial skin-draining lymph nodes. Melatonin also reduced cytokine-induced secretion of AD-related chemokines IP-10 and MCP-1 and decreased IL-4-induced cell proliferation in HaCaT cells. Melatonin seems to have an immunomodulatory effect on AD, with IP-10 as a possible target, and topical melatonin treatment is a potentially useful treatment for patients with AD.

Anti-Allergic and Anti-Inflammatory Effects of Neferine on RBL-2H3 Cells.

Mast cells play a very important role in skin allergy and inflammation, including atopic dermatitis and psoriasis. In the past, it was found that neferine has anti-inflammatory and anti-aging effects on the skin, but its effect on mast cells has not yet been studied in detail. In this study, we used mast cells (RBL-2H3 cells) and mouse models to study the anti-allergic and inflammatory effects of neferine. First, we found that neferine inhibits the degranulation of mast cells and the expression of cytokines. In addition, we observed that when mast cells were stimulated by A23187/phorbol 12-myristate-13-acetate (PMA), the elevation of intracellular calcium was inhibited by neferine. The phosphorylation of the MAPK/NF-κB pathway is also reduced by pretreatment of neferine. The results of in vivo studies show that neferine can improve the appearance of dermatitis and mast cell infiltration caused by dinitrochlorobenzene (DNCB). Moreover, the expressions of barrier proteins in the skin are also restored. Finally, it was found that neferine can reduce the scratching behavior caused by compound 48/80. Taken together, our results indicate that neferine is a very good anti-allergic and anti-inflammatory natural product. Its effect on mast cells contributes to its pharmacological mechanism.

Preliminary Results, Perspectives, and Proposal for a Screening Method of In Vitro Susceptibility of Prototheca Species to Antimicrotubular Agents.

Microorganisms belonging to the genus Prototheca are achlorophyllous microalgae, occasionally behaving as environmental pathogens that cause severe mastitis in milk cows, as well as localized or systemic infections in humans and animals. Among the different species belonging to the genus, Prototheca zopfii genotype 2 (recently reclassified as P. bovis) and P. blaschkeae are most commonly associated with bovine mastitis. To date, no pharmacological treatment is available to cure protothecal mastitis, and infected animals must be quarantined to avoid spreading the infection. The few antibiotic and antifungal drugs effective in vitro against Prototheca give poor results in vivo This failure is likely due to the lack of specificity of such drugs. As microalgae are more closely related to plants than to bacteria or fungi, an alternative possibility is to test molecules with herbicidal properties, in particular, antimicrotubular herbicides, for which plant rather than animal tubulin is the selective target. Once a suitable test protocol was set up, a panel of 11 antimicrotubular agents belonging to different chemical classes and selective for plant tubulin were tested for the ability to inhibit growth of Prototheca cells in vitro Two dinitroanilines, dinitramine and chloralin, showed strong inhibitory effects on P. blaschkeae at low micromolar concentrations, with half-maximal inhibitory concentrations (IC50) of 4.5 and 3 µM, respectively, while both P. zopfii genotype 1 (now reclassified as P. ciferrii) and P. bovis showed susceptibility to dinitramine only, to different degrees. Suitable screening protocols for antimitotic agents are suggested.

Ophiopogonin D ameliorates DNCB-induced atopic dermatitis-like lesions in BALB/c mice and TNF-α- inflamed HaCaT cell.

Atopic dermatitis (AD) can occur in both children and adults, and the symptoms include itching and eczema, which in turn cause patients to suffer. Ophiopogonin D (OP-D) is a steroidal glycoside from Radix Ophiopogon japonicus, which is well known as an effective anti-inflammatory herbal medicine in many Asian countries. In this study, we aimed to investigate the anti-inflammatory effects of OP-D, using an AD mouse model and inflamed HaCaT cells. Through a histopathological analysis, we were able to confirm the suppressive effects of OP-D on skin thickening and the mast cell activation in AD-like mouse back skin tissues stimulated by DNCB. In addition, we detected significant decreases in cytokine expression levels through multiplex assessment assays of the OP-D-treated mice blood. We observed the anti-inflammatory effect of OP-D in the spleen, causing weight loss in the spleen and in the mRNA expression levels related to diverse cytokines. In human keratinocytes inflamed by TNF-α, OP-D inhibited p38 and ERK protein activation and showed a reduction of NF-κB nuclear translocation. Furthermore, OP-D attenuated pro-inflammatory cytokine mRNA expressions in TNF-α-inflamed HaCaT cells. Accordingly, we came to the conclusion that OP-D is a potential natural drug which can be used in order to treat inflammatory skin diseases, such as AD.

Chaenomeles sinensis Koehne extract suppresses the development of atopic dermatitis-like lesions by regulating cytokine and filaggrin expression in NC/Nga mice.

Chaenomeles sinensis Koehne (CS) has been used in a traditional oriental medicine for treating throat diseases, anaphylaxis, viral infection, and inflammation. This study investigated the underlying mechanism of anti-allergic effect of CS. Leaves of CS plants were dried, powdered, and then underwent extraction with DMSO. Both ELISA and western blotting were performed to evaluate cytokine concentration and the expression and activation of filaggrin and JNK. Five-week-old female NC/Nga mice were used as an AD-like mouse model by treating them with 2,4-dinitrochlorobenzene (DNCB). The secretion of TARC, MCP-1, and IL-8 is increased by TNF-α and IFN-γ in HaCaT cells, and CS extract inhibited the increased production of TARC, MCP-1, and IL-8. TNF-α and IFN-γ suppressed filaggrin expression by activating JNK. CS extract recovered the expression of filaggrin decreased by TNF-α and IFN-γ by blocking the activation of JNK. In vivo experiment, CS administration reduced thickening of the epidermis and infiltration of inflammatory cells into the dermis as compared to DNCB treatment. Moreover, the decrease of filaggrin expression due to DNCB treatment was recovered by CS administration. The serum IgE level was decreased by CS treatment. The levels of IL-4, IL-5, IL-13 and eotaxin in mouse splenocytes increased after treatment with concanavalin A, and the secretions of IL-4, IL-5, IL-13 and eotaxin were lower in the CS-treated group than in the DNCB group. These results may contribute to the development of a CS-based drug for the treatment of atopic dermatitis.

Contact sensitizers trigger human CD1-autoreactive T-cell responses.

Allergic contact dermatitis is a primarily T-cell-mediated inflammatory skin disease induced by exposure to small molecular-weight haptens, which covalently bind to proteins. The abundance of cutaneous T cells that recognize CD1a antigen-presenting molecules raises the possibility that MHC-independent antigen presentation may be relevant in some hapten-driven immune responses. Here we examine the ability of contact sensitizers to influence CD1-restricted immunity. Exposure of human antigen-presenting cells such as monocyte-derived dendritic cells and THP-1 cells to the prototypical contact sensitizer dinitrochlorobenzene potentiated the response of CD1a- and CD1d-autoreactive T cells, which released a vast array of cytokines in a CD1- and TCR-dependent manner. The potentiating effects of dinitrochlorobenzene depended upon newly synthesized CD1 molecules and the presence of endogenous stimulatory lipids. Further examination of a broad panel of contact sensitizers revealed 1,4-benzoquinone, resorcinol, isoeugenol, and cinnamaldehyde to activate the same type of CD1-restricted responses. These findings provide a basis for the antigen-specific activation of skin-associated CD1-restricted T cells by small molecules and may have implications for contact sensitizer-induced inflammatory skin diseases.

Optimization of conditions for cadmium selenide quantum dot biosynthesis in Saccharomyces cerevisiae.

The biosynthesis of quantum dots has been explored as an alternative to traditional physicochemical methods; however, relatively few studies have determined optimal synthesis parameters. Saccharomyces cerevisiae sequentially treated with sodium selenite and cadmium chloride synthesized CdSe quantum dots in the cytoplasm. These nanoparticles displayed a prominent yellow fluorescence, with an emission maximum of approximately 540 nm. The requirement for glutathione in the biosynthetic mechanism was explored by depleting its intracellular content through cellular treatments with 1-chloro-2,4-dinitrobenzene and buthionine sulfoximine. Synthesis was significantly inhibited by both of these reagents when they were applied after selenite treatment prior to the addition of cadmium, thereby indicating that glutathione contributes to the biosynthetic process. Determining the optimum conditions for biosynthesis revealed that quantum dots were produced most efficiently at entry into stationary phase followed by direct addition of 1 mM selenite for only 6 h and then immediately incubating these cells in fresh growth medium containing 3 mM Cd (II). Synthesis of quantum dots reached a maximum at 84 h of reaction time. Biosynthesis of 800-µg g-1 fresh weight cells was achieved. For the first time, significant efforts have been undertaken to optimize each aspect of the CdSe biosynthetic procedure in S. cerevisiae, resulting in a 70% increased production.

6-Shogaol, an active compound of ginger, alleviates allergic dermatitis-like skin lesions via cytokine inhibition by activating the Nrf2 pathway.

Allergic dermatitis (AD) clinically presents with skin erythematous plaques, eruption, and elevated serum IgE, and T helper cell type 2 and 1 (Th2 and Th1) cytokine levels. 6-Shogaol [1-(4-hydroxy-methoxyphenyl)-4-decen-one], a pungent compound isolated from ginger, has shown anti-inflammatory effects, but its inhibitory effects on AD are unknown. The aim of this study was to examine whether 6-shogaol inhibits AD-like skin lesions and their underlying mechanism in vivo and in vitro. An AD-like response was induced by tumor necrosis factor-α (TNF-α)+IFN-γ in human keratinocytes or by 2,4-dinitrochlorobenzene (DNCB) in mice. In vivo, 6-shogaol inhibited the development of DNCB-induced AD-like skin lesions and scratching behavior, and showed significant reduction in Th2/1-mediated inflammatory cytokines, IgE, TNF-α, IFN-γ, thymus and activation-regulated chemokine, IL-1, 4, 12, and 13, cyclooxygenase-2, and nitric oxide synthase levels. In vitro, 6-shogaol inhibited reactive oxygen species (ROS) and mitogen-activated protein kinases (MAPKs) signaling, and increased the levels of total glutathione, heme oxygenase-1, and quinone 1 via nuclear factor erythroid 2 related factor 2 (Nrf2) activation. 6-Shogaol can alleviate AD-like skin lesions by inhibiting immune mediators via regulating the ROS/MAPKs/Nrf2 signaling pathway, and may be an effective alternative therapy for AD.

Glutathione protects Candida albicans against horseradish volatile oil.

Horseradish essential oil (HREO; a natural mixture of different isothiocyanates) had strong fungicide effect against Candida albicans both in volatile and liquid phase. In liquid phase this antifungal effect was more significant than those of its main components allyl, and 2-phenylethyl isothiocyanate. HREO, at sublethal concentration, induced oxidative stress which was characterized with elevated superoxide content and up-regulated specific glutathione reductase, glutathione peroxidase, catalase and superoxide dismutase activities. Induction of specific glutathione S-transferase activities as marker of glutathione (GSH) dependent detoxification was also observed. At higher concentration, HREO depleted the GSH pool, increased heavily the superoxide production and killed the cells rapidly. HREO and the GSH pool depleting agent, 1-chlore-2,4-dinitrobenzene showed strong synergism when they were applied together to kill C. albicans cells. Based on all these, we assume that GSH metabolism protects fungi against isothiocyanates.

Glutathione and its related enzymes in the gonad of Nile Tilapia (Oreochromis niloticus).

Glutathione (GSH) concentration, the activity of its metabolizing enzymes, glutathione transferase (GST), glutathione peroxidase (GPx), glutathione reductase (GR), and the antioxidant enzyme catalase (CAT) in O. niloticus ovary and testis were examined. GSH concentration of O. niloticus testis exhibited high concentration (129 ± 21 nmol/g tissue) compared with GSH concentration (49.2 ± 8.3 nmol/g tissue) in the ovary. GST, GPx, GR, and CAT activities of O. niloticus testis exhibited high values compared with their corresponding values in ovary homogenates. However, protein concentration in ovary homogenates exhibited higher values (175 ± 40.6 mg) compared with testis homogenates (27.1 ± 3.7 mg). O. niloticus ovary was less effective in excretion of xenobiotices compared with the testis, where its function is mainly in increasing the protein content of the eggs; however, in O. niloticus testis, the glutathione cycle operated in accelerated way in the direction of reduced GSH production in order to protect the maturation stages in a save way. A simple reproducible procedure for the purification of GST from O. niloticus ovary was established. The enzymes proved to be homogenous as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and its molecular weight was calculated to be 25.1 kDa. GST of O. niloticus ovary exhibited maximum activity at pH 7.5. The Michaelis-Menten constant (K(m)) of the purified ovary GST for GSH and CDNB was 0.076 mM and 1.0 mM, respectively. Cibacron blue was the most potent inhibitor of ovary GST activity (IC50 value, concentration of inhibitor that will give 50% inhibition, equal 0.002 µM). The specific activity of GST toward different electrophilic substrates was determined. GST activity toward benzyl isothiocyanate was the highest compared with phenethyl isothiocyanate and allyl isothiocyanate.

Coordinated induction of GST and MRP2 by cAMP in Caco-2 cells: Role of protein kinase A signaling pathway and toxicological relevance.

The cAMP pathway is a universal signaling pathway regulating many cellular processes including metabolic routes, growth and differentiation. However, its effects on xenobiotic biotransformation and transport systems are poorly characterized. The effect of cAMP on expression and activity of GST and MRP2 was evaluated in Caco-2 cells, a model of intestinal epithelium. Cells incubated with the cAMP permeable analog dibutyryl cyclic AMP (db-cAMP: 1,10,100 µM) for 48 h exhibited a dose-response increase in GST class α and MRP2 protein expression. Incubation with forskolin, an activator of adenylyl cyclase, confirmed the association between intracellular cAMP and upregulation of MRP2. Consistent with increased expression of GSTα and MRP2, db-cAMP enhanced their activities, as well as cytoprotection against the common substrate 1-chloro-2,4-dinitrobenzene. Pretreatment with protein kinase A (PKA) inhibitors totally abolished upregulation of MRP2 and GSTα induced by db-cAMP. In silico analysis together with experiments consisting of treatment with db-cAMP of Caco-2 cells transfected with a reporter construct containing CRE and AP-1 sites evidenced participation of these sites in MRP2 upregulation. Further studies involving the transcription factors CREB and AP-1 (c-JUN, c-FOS and ATF2) demonstrated increased levels of total c-JUN and phosphorylation of c-JUN and ATF2 by db-cAMP, which were suppressed by a PKA inhibitor. Co-immunoprecipitation and ChIP assay studies demonstrated that db-cAMP increased c-JUN/ATF2 interaction, with further recruitment to the region of the MRP2 promoter containing CRE and AP-1 sites. We conclude that cAMP induces GSTα and MRP2 expression and activity in Caco-2 cells via the PKA pathway, thus regulating detoxification of specific xenobiotics.

How important are glutathione and thiol reductases to oyster hemocyte function?

Bivalves are animals with worldwide distribution. Although they play key roles in economic activities, human feeding and environmental studies, there is a considerable lack of knowledge about the relationship between their immune system and antioxidant defenses. Here, we performed an in vitro experiment where Crassostrea gigas hemocytes were exposed to the electrophilic compound 1-chloro-2,4-dinitrobenzene (CDNB, 0.1-50 µM) for one hour. CDNB treatment clearly disturbed thiol homeostasis, causing a concentration-dependent decrease in the glutathione (GSH) content and a decrease in the activity of two thiol reductases, glutathione reductase (GR - 2.5 and 50 µM CDNB) and thioredoxin reductase (TrxR - only 50 µM CDNB). The MTT reduction assay showed that none of the CDNB concentrations tested significantly altered cell viability. However, there was a decrease in the hemocyte's ability to uptake the neutral red dye, which indicates lysosomal impairment (≥12.5 µM CDNB). Cellular immunocompetence was further investigated and, despite the lower GSH content, GR activity and impairment in lysosome integrity, hemocyte functions (adhesion capacity, phagocytosis of latex beads and laminarin-induced ROS production) were preserved in the 2.5 and 12.5 µM CDNB treatments. These results suggest a minor importance of thiol pools and GR activity in C. gigas hemocyte's immunocompetence, in an in vitro acute exposure model. The 50 µM CDNB treatment, however, significantly compromised all the measured hemocyte functions. This response was associated with TrxR inhibition, increased lysosome impairment, decreased GSH content, and lower GR activity. Therefore, it seems that TrxR may be particularly important for the hemocyte function, or, alternatively, it is only affected when a deeply aggravated scenario in thiol homeostasis is set up. Such findings point out the need for further studies towards a better understanding of antioxidant and immune defenses interactions in bivalve cellular systems.